Leishmania Patents (Class 435/258.3)
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Publication number: 20140356960Abstract: The present invention relates to a nucleic acid containing at least one homing endonuclease site (HE) and at least one restriction enzyme site (X) wherein the HE and X sites are selected such that HE and X result in compatible cohesive ends when cut by the homing endonuclease and restriction enzyme, respectively, and the ligation product of HE and X cohesive ends can neither be cleaved by the homing endonuclease nor by the restriction enzyme. Further subject-matter of the present invention relates to a vector comprising the nucleic acid of the present invention, host cells containing the nucleic acid and/or the vector; a kit for cloning and/or expression of multiprotein complexes making use of the vector and the host cells, a method for producing a vector containing multiple expression cassettes, and a method for producing multiprotein complexes.Type: ApplicationFiled: April 25, 2014Publication date: December 4, 2014Inventor: Imre Berger
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Patent number: 8877213Abstract: Targeted disruption of the centrin gene leads to attenuation of growth in the Leishmania. Preferred embodiments of the invention provide attenuated strains of Leishmania useful for the preparation of immunogenic preparations including vaccines against a disease caused by infection with a virulent Leishmania strain and as tools for the generation of immunological and diagnostic reagents. Other preferred embodiments provide related immunogenic compositions, methods of generating an immune response, methods for producing a vaccine, and methods of forming attenuated strains of Leishmania.Type: GrantFiled: November 15, 2010Date of Patent: November 4, 2014Assignees: The United States of America as Represented by the Secretary of the Department of Health and Human Services, Indian Council of Medical ResearchInventors: Hira L. Nakhasi, Angamuthu Selvapandiyan, Alain Debrabant, Robert C. Duncan, Poonam Salotra, Gannavaram Sreenivas
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Publication number: 20140120156Abstract: The present application relates to methods of producing exosomes. The application also provides a method for preparing a protein composition comprising culturing an exosome-producing cell expressing a Nef-fusion protein comprising a Nef-derived peptide fused to a protein of interest; isolating exosomes from the exosome-producing cell culture; and purifying the protein of interest from the isolated exosomes. The application further discloses compositions that comprise exosomes containing the Nef-fusion protein, as well as methods of using the Nef-fusion protein and exosomes containing the Nef-fusion protein.Type: ApplicationFiled: September 6, 2013Publication date: May 1, 2014Inventors: Vincent Craig BOND, Michael POWELL, MingBo HUANG, Syed ALI
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Publication number: 20140004181Abstract: The present application relates to methods of producing exosomes. The application also provides a method for preparing a protein composition comprising culturing an exosome-producing cell expressing a Nef-fusion protein comprising a Nef-derived peptide fused to a protein of interest; isolating exosomes from the exosome-producing cell culture; and purifying the protein of interest from the isolated exosomes. The application further discloses compositions that comprise exosomes containing the Nef-fusion protein, as well as methods of using the Nef-fusion protein and exosomes containing the Nef-fusion protein.Type: ApplicationFiled: September 6, 2013Publication date: January 2, 2014Applicant: Morehouse School of MedicineInventors: Vincent Craig BOND, Michael Powell, Ming Bo Huang, Syed Ali
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Publication number: 20130164329Abstract: The present invention relates to compositions and methods for isolating cells devoid of unwanted viral contaminants, and to methods for preparing a virus stock substantially devoid of viral contaminants. Virus stocks, cells, and immunogenic reagents produced using such methods are also provided.Type: ApplicationFiled: February 16, 2011Publication date: June 27, 2013Applicant: Alnylam Pharmaceuticals, Inc.Inventors: Anthony Rossomando, John M. Maraganore, Stuart Pollard, Muthiah Manoharan
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Publication number: 20130017224Abstract: Nucleic acid molecules comprising a nucleotide sequence encoding RANTES and fragments and variants thereof are disclosed. Additionally, nucleic acid molecules and compositions comprising the nucleotide sequence encoding RANTES and fragments and variants thereof in combination with nucleic acid sequences encoding immunogens are provided. Recombinant viral vectors comprising the nucleotide sequence encoding RANTES and fragments and variants thereof with or without a nucleic acid sequence encoding immunogens are also provided as are live attenuated pathogens comprising a nucleotide sequence encoding RANTES and fragments and variants thereof. Methods of modulating immune responses and of inducing an immune response against an immunogen are also disclosed.Type: ApplicationFiled: February 8, 2011Publication date: January 17, 2013Inventors: David B. Weiner, Jean D. Boyer, Michele Kutzler
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Publication number: 20120288524Abstract: The present invention provides a composition for delivering a protein vaccination candidate to a mammalian subject having a Leishmania transfected for expressing a cDNA sequence for encoding the protein vaccination candidate, and the Leishmania containing a photosensitizer.Type: ApplicationFiled: May 10, 2012Publication date: November 15, 2012Applicant: ROSALIND FRANKLIN UNIVERSITY OF MEDICINE AND SCIENCEInventors: Kwang-Poo CHANG, Sujoy DUTTA
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Publication number: 20120289689Abstract: The present invention relates to a recombinant process for the production of truncated or mutated dextransucrases while conserving the enzymatic activity or their specificity in the synthesis of the ?-1,6 bonds. The present invention relates to nucleic acid sequences of truncated or mutated dextransucrases, vectors containing the nucleic acid sequences and host cells transformed by sequences encoding truncated or mutated dextransucrases. In another aspect, the invention concerns a method for producing, in a recombinant manner, truncated or mutated dextransucrases which conserve their enzymatic activity or which conserve their specificity in the synthesis of ?-1,6 bonds and can produce, from saccharose, dextrans with high molar mass and modified rheological properties compared with the properties of dextran obtained with the native enzyme and isomalto-oligosaccharides with a controlled molar mass and dextrans.Type: ApplicationFiled: August 2, 2012Publication date: November 15, 2012Inventors: Pierre F. Monsan, Magali Remaud-Simeon, Gabrielle Potocki-Veronese, Claire Moulis
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Patent number: 7993917Abstract: Methods for the diagnosis of visceral, cutaneous and canine leishmaniasis in a subject suspected of being infected with the parasitic protozoa Leishmania is disclosed. Disclosed are antibody-capture enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies to Leishmania parasite soluble antigens and antigen-capture ELISAs for the detection of Leishmania parasite soluble antigens in host samples. Also disclosed are immunodiagnostic kits for the detection of Leishmania parasite circulating antigens or IgM and IgG antibodies in a sample from subject having visceral, cutaneous or canine leishmaniasis. In these methods and kits, detection may be done photometrically or visually. The methods and kits also allow the visualization of Leishmania amastigotes or promastigotes in a sample.Type: GrantFiled: August 20, 2009Date of Patent: August 9, 2011Assignee: The United States of America as represented by the Secretary of the ArmyInventor: Samuel K. Martin
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Publication number: 20110104064Abstract: Non-native synthetic peptide cyclized by means of a disulphide bridge, characterized by the amino acid sequence (E34P) that follows (SEQ ID No. 1): E-D-E-H-K-G-K-Y-C-R-L-G-N-D-C-R-T-T-E-P-T-T-T-A-T-P-R-G-T-P-T-P-A-P and a complex of non-native synthetic peptides that is intended to induce and characterize the prevention and/or treatment of conditions in mammals, the protective immunity of which depends on the stimulation of Th1 type lymphocytes, and, in particular, on a state of delayed hyperstimulation, containing: —said peptide (E34P), —the following sequence (A16E) of 16 amino acids (SEQ ID No. 2) or derivatives (structural analogues) thereof: A-A-R-C-A-R-C-R-E-G-Y-S-L-T-D-E —the following sequence (A16G) of 16 amino acids (SEQ ID No. 3) or derivatives (structural analogues) thereof: A-A-S-S-T-P-S-P-G-S-G-C-E-V-D-G, —and an adjuvant which preferably induces a cell-mediated response.Type: ApplicationFiled: June 18, 2009Publication date: May 5, 2011Applicant: ORIDAN INC.Inventors: Serge Vicens, Grégory Hottin
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Publication number: 20110081383Abstract: Targeted disruption of the centrin gene leads to attenuation of growth in the Leishmania. Preferred embodiments of the invention provide attenuated strains of Leishmania useful for the preparation of immunogenic preparations including vaccines against a disease caused by infection with a virulent Leishmania strain and as tools for the generation of immunological and diagnostic reagents. Other preferred embodiments provide related immunogenic compositions, methods of generating an immune response, methods for producing a vaccine, and methods of forming attenuated strains of Leishmania.Type: ApplicationFiled: November 15, 2010Publication date: April 7, 2011Applicants: The Gov. of the United States as Represented by the Secretary, Dept. of Health and Human Services, Institute of Pathology (ICMR)Inventors: Hira L. Nakhasi, Angamuthu Selvapandiyan, Alain Debrabant, Robert C. Duncan, Poonam Salotra, Gannavaram Sreenivas
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Patent number: 7732188Abstract: The present invention relates to a protozoan strain of the family of the Trypanosomatidae the virulence of which is lower than that of the corresponding wild-type strain, characterized in that at least one copy of the SIR2 gene present in the genome of the strain is inactivated.Type: GrantFiled: October 25, 2004Date of Patent: June 8, 2010Assignees: Institut de Recherche pour le Developpement, Universidade Do PortoInventors: Anabela Cordeira Da Silva, Ali Ouaissi, Denis Sereno, Baptiste Vergnes
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Patent number: 7700121Abstract: The invention relates to the field of combating leishmaniases. Said invention results from the isolation, from wild isolates of Leishmania major, of a protein-coding gene known as LmPDI which has two regions that are identical to the sequence (Cys-Gly-His-Cys) of the potential active site of the protein disulphide isomerases (PDI). The LmPDI protein is predominantly expressed in the most virulent isolates of the parasite. Said protein forms a novel therapeutic target for developing anti-leishmaniasis medicaments and a novel element that can be used in the composition of immunogenic, and possibly vaccinating, preparations which are intended to protect a human or animal host against Leishmania.Type: GrantFiled: December 12, 2003Date of Patent: April 20, 2010Assignees: Institut Pasteur, Institut Pasteur de TunisInventors: Yosser Ben Achour, Mehdi Chenik, Hechmi Louzir, Koussay Dellagi
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Publication number: 20090280536Abstract: The invention provides a novel system for the tunable expression of nucleic acids encoding e.g., polypeptides such as recombinant proteins in prokaryotic systems. The system is based on the ability of T7 lysozyme (T7Lys) to inhibit the activity of T7RNAP. Expression of T7Lys can be continuously adjusted as its expression is under the control of a promoter whose activity can be titrated. The invention provides a host cell capable of expressing T7 RNA polymerase, the host cell comprising a first nucleic acid having a T7 lysozyme gene or a T7 lysozyme variant gene and a tunable promoter for controlling the expression of the T7 lysozyme gene. It also provides a host cell further comprising a second nucleic acid having a T7 promoter operably linked to a nucleic acid sequence encoding a target polypeptide, whereby expression of the target polypeptide is tuned via controlling the expression of the T7 lysozyme gene.Type: ApplicationFiled: February 27, 2009Publication date: November 12, 2009Inventors: Jan Willem DE GIER, Samuel Wagner
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Publication number: 20090214595Abstract: The invention relates to an expression vector of a gene coding for an antigenic protein of Leishmania promastigote, characterised in that it comprises a PSA gene insert in opposite orientation. The invention can be applied to the development of mutants under-expressing, or no longer expressing, genes coding for an antigenic protein of Leishmania promastigote, and to the therapeutic and/or vaccine-oriented uses thereof.Type: ApplicationFiled: February 10, 2006Publication date: August 27, 2009Applicant: INSTITUT DE RECHERCHE POUR LE DEVELOPPEMENT (IRD)Inventors: Jean-Loup Lemesre, Philippe Holzmuller, Rachel Bras-Goncalves
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Publication number: 20090169554Abstract: The present invention relates to new proteins of Leishmania major and to therapeutical and diagnostic applications thereof. More particularly, the present invention relates to excreted/secreted polypeptides and polynucleotides encoding same, compositions comprising the same, and methods of diagnosis, vaccination and treatment of Leishmaniasis.Type: ApplicationFiled: April 7, 2006Publication date: July 2, 2009Inventors: Mehdi Chenik, Sami Lakhal, Hechmi Louzir, Koussay Dellagi
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Patent number: 7456018Abstract: Human hepatoma cell lines are provided that comprises a set of cells clonally derived from a cancerous human liver cell, in which each cell of the set expresses a receptor having an affinity for binding a virus of the Flaviviridae genus and a virus of the Hepadnaviridae genus to enable infection by viruses of both genus in native forms, and in which each cell of the set exhibits susceptibility to infection by a hepatotropic parasite of the Leishmania genus in a native form. Various compositions and methods relating to diagnostic, therapeutic, and prophylactic embodiments are also provided.Type: GrantFiled: July 8, 2002Date of Patent: November 25, 2008Inventors: Philippe Gripon, Christiane Guguen-Guillouzo, Christian Trepo, Sylvie Rumin
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Patent number: 7452721Abstract: Methods for the diagnosis of visceral, cutaneous and canine leishmaniasis in a subject suspected of being infected with the parasitic protozoa Leishmania is disclosed. Disclosed are antibody-capture enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies to Leishmania parasite soluble antigens and antigen-capture ELISAs for the detection of Leishmania parasite soluble antigens in host samples. Also disclosed are immunodiagnostic kits for the detection of Leishmania parasite circulating antigens or IgM and IgG antibodies in a sample from subject having visceral, cutaneous or canine leishmaniasis. In these methods and kits, detection may be done photometrically or visually. The methods and kits also allow the visualization of Leishmania amastigotes or promastigotes in a sample.Type: GrantFiled: November 18, 2004Date of Patent: November 18, 2008Assignee: The United States of America as represented by the Secretary of the ArmyInventor: Samuel K. Martin
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Publication number: 20080241883Abstract: Compositions and methods comprising recombinant expression vector elements (rEVEs) to enhance the level of expression of recombinant proteins are described. Other compositions and methods for lowering, substantially suppressing, or essentially silencing expression of a recombinant protein are also described.Type: ApplicationFiled: March 28, 2008Publication date: October 2, 2008Inventors: Wendy R. Gion, Gerald R. Carson, Hong Gao, Yune Z. Kunes
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Publication number: 20080026467Abstract: The invention relates to nucleic acid constructions, characterized in that they comprise nucleic acids which are isolated in the sense position and which are capable of coding for an immunogenic protein of promastigotes or amastigotes of Leishmania, said nucleic acids responding to one of the sequences SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 et SEQ ID No. 11 and coding for a protein respectively exhibiting a sequence SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10 et SEQ ID No. 12. The invention can be used for over-expression of the genes of Leishmania coding for an excretion/secretion antigen.Type: ApplicationFiled: November 19, 2004Publication date: January 31, 2008Inventors: Jean-Loup Lemesre, Mireille Cavaleyra, Denis Sereno, Philippe Holzmuller
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Patent number: 7261887Abstract: Methods for delivering potentially therapeutic or prophylactic protein and peptide agents to mammalian cells are provided. The agents are delivered by mutant trypanosomatid protozoa that have been genetically manipulated to code for such protein or peptide agents. The mutant protozoa additionally lack certain enzymes within the heme biosynthetic pathway, making the mutants susceptible to porphyria and eventual lysis.Type: GrantFiled: November 12, 2002Date of Patent: August 28, 2007Assignee: Rosalind Franklin University of Medicine and ScienceInventors: Kwang-Poo Chang, Shigeru Sassa, Jerome F. Sah, Hiroya Ito, Bala Krishna Kolli, Daniel A. Peterson
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Patent number: 7238347Abstract: The present invention discloses the use of a mutant Leishmania as a suicidal vaccine wherein the mutant Leishmania is responsive to external signals to become porphyric and commit suicidal cytolysis. The mutant can be selected from natural Leishmania species or constructed by genetic engineering.Type: GrantFiled: March 28, 2005Date of Patent: July 3, 2007Assignee: Rosalind Franklin University of Medicine and ScienceInventors: Kwang-Poo Chang, Bala Krishna Kolli, Shigeru Sassa
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Patent number: 6955890Abstract: A method for the identification and treatment of pathogenic microorganism infections by inhibiting one or more enzymes in a metabolic pathway by inhibiting the conversion of substrate to produce the penultimate or ultimate product particularly by inhibiting the activity of one or more of the enzymes in the pathwayType: GrantFiled: May 10, 2002Date of Patent: October 18, 2005Assignee: Pyro Pharmaceuticals, Inc.Inventor: Alan M. Schechter
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Patent number: 6890542Abstract: The present invention relates to the use of a live mutant Leishmania in the preparation of a vaccine and to vaccine formulations for use in immunizing mammals, such as dogs and/or humans. The mutant Leishmania comprises at least one defective cysteine proteinase gene.Type: GrantFiled: April 24, 2003Date of Patent: May 10, 2005Assignee: University Court of the University of GlasgowInventors: Jeremy Charles Mottram, Graham Herbert Coombs
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Patent number: 6855537Abstract: Strains of Leishmania and other macrophage-infecting parasites are provided which express the GM-CSF gene which are useful in treating hosts infected by the parasite and in protecting hosts against disease caused by infection of hosts by parasites. The parasites are reduced in their ability to infect or survive in macrophages and hence are attenuated. At least one gene of the parasite contributing to the virulence thereto may be functionally disabled. The attenuated strains may be used for administration to a host (a) to treat a host infected by Leishmania or (b) to confer protection against disease caused by a virulent Leishmania strain, or as a diagnostic reagent.Type: GrantFiled: September 16, 1997Date of Patent: February 15, 2005Assignee: Aventis Pasteur LimitedInventors: Barbara Papadopoulu, Marc Ouellette, Martin Olivier
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Patent number: 6652847Abstract: Attenuated strains of Leishmania are provided in which at least one gene contributing to virulence of the strain and expressed in both the promastigote and amastigote forms of the strain is functionally disabled, such as, by deleting at least a portion of the gene or by mutagenesis of the gene. The attenuated strain may be used for administration to a host to confer protection against disease caused by a virulent Leishmania strain or as a diagnostic reagent.Type: GrantFiled: July 17, 2000Date of Patent: November 25, 2003Assignee: Universite LavalInventors: Barbara Papadopoulou, Marc Ouellette, Martin Olivier
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Publication number: 20030138461Abstract: The process of the invention comprises the implementation of axenic conditions, with use of a liquid single-phase culture medium. For obtaining the amastigote forms, this medium is buffered at a pH of 5.5 to 6.5 and has an osmolarity of at least 400 milliosmoles/kg of liquid, and in particular 400 to 550 milliosmoles/kg of liquid. For obtaining promastigote forms, this medium is buffered at a pH of 7 to 7.5 and has an osmolarity of at least 300 milliosmoles/kg of liquid. This process allows the adaptation and culture in vitro of different stages of tissular parasites, such as leishmanias and T. cruzi or also hematoprotozoa.Type: ApplicationFiled: October 1, 2002Publication date: July 24, 2003Applicant: Institut Francais de Recherche Scientifique Pour Le Developpement en Cooperation (ORSTOM)Inventor: Jean-Loup Lemesre
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Publication number: 20030059439Abstract: The present invention relates to the use of the major OprI lipoprotein of Pseudomonas aeruginosa to elicit a Type-1 immune response towards a heterologous antigen. The invention relates specifically to the use of OprI—antigen fusion proteins to elicit the Type-1 response. More particularly, the present invention is directed to pharmaceutical formulations comprising OprI and/or OprI fusion proteins, optionally together with a suitable excipient, to stimulate the Th1 dependent, cellular immune response.Type: ApplicationFiled: August 16, 2002Publication date: March 27, 2003Inventors: Hilde Revets, Pierre Cornelis, Patrick De Baetselier
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Publication number: 20020155562Abstract: The present invention describes an enzyme showing glutathionylspermidine synthetase-activity and being distinct from known enzymes with similar activities in several physicochemical parameters, a novel process to isolate said enzyme from Crithidia fasciculata, tools for the production thereof in genetically transformed organisms, and its use as a molecular target for the discovery of trypanocidal drugs.Type: ApplicationFiled: September 17, 2001Publication date: October 24, 2002Applicant: Leopold FloheInventors: Leopold Flohe, Kerstin Koenig, Ulrich Menge
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Publication number: 20020147321Abstract: Chimeric gene formed by the DNA sequences that encode the antigenic determinants of four proteins of L. infantum, useful for the serological diagnosis of canine Leishmaniosis and protein obtained, that consists of the prior employment of a cloning strategy. The patent describes the intermediate products generated during the process. A clone is achieved expressed in the protein rLiPO-Ct-Q (pPQI). To this initial vector, by means of the use of suitable restriction targets, DNA fragments are sequentially added that are encoded in other proteins and after each cloning step the correct orientation of each one of the inserts reduces the size of the expression products, the complete nucleotide sequence of the final pPQV clone being determined. A polypeptide is obtained with a molecular mass of 38 kD and with an isoelectric point of 7.37.Type: ApplicationFiled: February 21, 2001Publication date: October 10, 2002Inventors: Carlos Alonso Bedate, Jose Maria Requena Rolania, Manuel Soto Alvarez
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Patent number: 6458581Abstract: The present invention provides a method for carrying out in vitro the complete developmental sequence culture of tissular parasites, which includes culturing the parasites in a totally defined culture medium which is an axenic monophasic liquid culture medium, free of serum and free of serum-derived or cell-derived macromolecules, proteins and peptides. For obtaining amastigote forms, this medium is buffered at a pH of 5.5 to 6.5 and has an osmolarity of at least 400 milliosmoles/kg of liquid. For obtaining promastigote forms, the medium is buffered at a pH of 7 to 7.5 and has an osmolarity of at least 300 milliosmoles/kg liquid. Application to the in vitro culture of different stages of tissular parasites such as Leishmania, T. cruzi, and hamatoprotozoa is also provided.Type: GrantFiled: November 9, 1995Date of Patent: October 1, 2002Assignee: Institut francais de recherche scientifique pour le developpement en Cooperation (ORSTOM)Inventor: Jean-Loup Lemesre
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Patent number: 6426203Abstract: Attenuated strains of Leishmania are provided in which at least one gene contributing to virulence of the strain and expressed in both the promastigote and amastigote forms of the strain is functionally disabled, such as, by deleting at least a portion of the gene or by mutagenesis of the gene. The attenuated strain may be used for administration to a host to confer protection against disease caused by a virulent Leishmania strain or as a diagnostic reagent.Type: GrantFiled: July 17, 2000Date of Patent: July 30, 2002Assignee: Universite LavalInventors: Barbara Papadopoulou, Marc Ouellette, Martin Olivier
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Patent number: 6331304Abstract: Strains of Leishmanda and other macrophage-infecting parasites are provided which express the GM-CSF gene which are useful in treating hosts infected by the parasite and in protecting hosts against disease caused by infection of hosts by parasites. The parasites are reduced in their ability to infect or survive in macrophages and hence are attenuated. At least one gene of the parasite contributing to the virulence thereto may be functionally disabled. The attenuated strains may be used for administration to a host (a) to treat a host infected by Leishmania or (b) to confer protection against disease caused by a virulent Leishmania strain, or as a diagnostic reagent.Type: GrantFiled: September 13, 1996Date of Patent: December 18, 2001Assignee: Universite LavalInventors: Barbara Papadopoulou, Marc Ouellette, Martin Olivier
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Patent number: 6291217Abstract: The present invention describes an enzyme showing glutathionylspermidine synthetase-activity and being distinct from known enzymes with similar activities in several physicochemical parameters, a novel process to isolate said enzyme from Crithidia fasciculata, tools for the production thereof in genetically transformed organisms, and its use as a molecular target for the discovery of trypanocidal drugs.Type: GrantFiled: June 11, 1999Date of Patent: September 18, 2001Inventors: Leopold Flohé, Kerstin Koenig, Ulrich Menge
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Patent number: 6261523Abstract: An adjustable-volume, sealed chemical-solution-confinement vessel for enclosing the inner bottom surface of the chemical-solution-confinement vessel within a sealed chamber. The chemical-solution-confinement vessel comprises a well-shaped base into which a complementarily-shaped lid is inserted. The lid can be inserted to a first position, in order to create a small volume sealed chamber above the inner bottom surface of the chemical-solution-confinement vessel, and can be retracted to a second position in order to create a larger volume sealed chamber above the inner bottom surface of the chemical-solution-confinement vessel. The lid may contain a port or aperture for introducing a fluid or material into the sealed chamber.Type: GrantFiled: April 27, 1999Date of Patent: July 17, 2001Assignee: Agilent Technologies Inc.Inventor: Carol T. Schembri
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Patent number: 6228649Abstract: Homozygous gene replacement can be created in unicellular diploid organisms by individually targeting each allele of a gene with genetic constructs containing two different and independent selectable markers. Selection for both markers indicates replacement of both alleles of the gene, or portion thereof. The method can be used to study gene function in these organisms and to create mutant organisms such as attenuated strains of parasitic protozoans for use in live vaccines.Type: GrantFiled: January 22, 1999Date of Patent: May 8, 2001Assignee: President and Fellows of Harvard CollegeInventors: Stephen M. Beverley, Angela K. Cruz